A serious problem associated with cancer chemotherapy is the development of multidrug-resistant (MDR) tumor cells during the course of treatment. An important mechanism of acquiring the MDR phenotype in mammalian cells is the enhanced expression of a membrane glycoprotein, termed Pgp (Reference 1). Pgp, having a molecular weight of 170 kDa, is coded by the MDR1 gene.
Pgp functions as an energy-dependent multidrug membrane transporter that rapidly extrudes a variety of hydrophobic antitumor drugs from target cancer cells, and thereby prevents the drugs from exerting cytotoxic effects. Initial physiological and pharmacological studies with multidrug-resistant mutant cell lines correlated resistance to reduced accumulation of drugs within the cell due to increased efflux or decreased influx (1). Because the efflux pump is an ATP-dependent transport system (2), agents that are good inhibitors of ATP-dependent drug transport should inhibit the efflux of hydrophobic drugs from resistant cells and increase intracellular accumulation. Accordingly, a variety of agents have been reported to overcome or at least partially circumvent MDR (3).
First-generation modulators originally were developed for other therapeutic indications (4). Included in this category are calcium channel blockers, such as verapamil (5), the immunosuppressive agent cyclosporin A (6), analogues of the anti-hypertensive reserpine and yohimbine (7), the neuroleptic agent, trifluroperazine (4), and antiestrogens, such as tamoxifen (1). Second-generation modulators were developed that lacked the pharmacological activities of the first-generation compounds and usually possessed higher affinity for Pgp. These agents include the R isomer of verapamil (8), a nonimmunosuppressive analogue of cyclosporin D, SDZ PSC-833 (6), and others such as MS-209 (9), S-9788 (10), GF120918 (11), and LY335979 (4).
While many of these pharmacological agents have been found to completely overcome drug resistance in in vitro models, the number of reports showing such phenomena in in vivo systems is more limited (12–15). The lack of in vivo activity of chemosensitizers results mainly from problems associated with maintaining active doses without causing serious side effects (16). Thus, clinical phase I and phase I/II studies often have been disappointing because of limited tolerance to prototype MDR inhibitors by themselves, which precluded attainment of potentially active levels in patients (11, 17).
For example, full reversion of MDR by verapamil requires a concentration of approximately 10 μM in most cell culture models, whereas plasma levels above 1 μM result in atrioventricular blocks (5, 18). Immunosuppressive effects and nephrotoxicity limit the clinical usefulness of cyclosporin A (6), and toxicities, such as cerebellar ataxia and hyperbilirubinemia, are caused by SDZ PSC-833 (19, 20). Also, cyclosporin A, verapamil, and SDZ PSC-833 have profound effects on the pharmacokinetics of doxorubicin, etoposide, and other oncolytic drugs (4).
As recently reported (21), the MDR modulator valspoday is being evaluated in phase III, randomized trials for the treatment of AML, multiple myeloma, and ovarian cancer. Also, Ontogen Corporation has announced completion of a phase I study conducted with the MDR-reversing agent OC 144-093 (22) and a second phase I study to examine oral administration is underway.
One part of a natural product drug discovery program involves monitoring the potential of plant extracts to reverse multiple drug resistance. Standard cell survival assays are used to determine the dose of plant extracts or compounds required to inhibit cell growth by 50% (IC50) with drug-sensitive human epidermoid carcinoma parental KB-3 cells and Pgp-associated multidrug-resistant KB-V1 cells. To investigate the potential of plant extracts or compounds to reverse multidrug-resistance, KB-V1 cells are treated with different concentrations of plant extracts or compounds in the presence (1 μg/ml) or absence of vinblastine. This concentration of vinblastine is lethal to KB-3 cells, but does not affect the growth of KB-V1 cells. Therefore, KB-3 cells serve as a control to differentiate between nonspecific cytotoxicity and selective MDR antagonism.
The assay employs 96-well microtiter plate technology, and over 3,000 different plant extracts have been tested. Using the model for bioassay-guided isolation of active principles, four moderate inhibitors of MDR have been identified: coronaridine, conoduramine, voacamine, and (−)-roemerine (23, 24).
The present invention, therefore, is directed to additional multidrug resistance inhibitors that are isolated from Erythroxylum pervillei and overcome the problem associated with prior inhibitors.